Potentiation of antifungal activity of amphotericin B by azithromycin againstAspergillus species

The potential role of azithromycin in combination with amphotericin B against 25 clinical isolates ofAspergillus was assessed. The MIC of amphotericin B was 1 g/ml for 44% of the isolates, 0.5 g/ml for 48%, and 0.25 g/ml for 8%. All isolates were resistant to azithromycin. Synergism, defined as a twofold reduction in the MIC of both drugs upon combination, was demonstrated between amphotericin B and azithromycin for all 25 isolates. To prove that azithromycin exerts its antifungal effect by inhibiting protein synthesis, we studied [³⁵S]-methionine incorporation into protein inone Aspergillus isolate. Neither amphotericin B at 0.125 g/ml (fourfold below its MIC) nor azithromycin at 16 g/ml ( 16-fold below its MIC) had any effect on protein synthesis when tested alone. Upon combination, however, a 68% inhibition in protein synthesis was evident by the inhibition of [³⁵S]-methionine incorporation.     

Provisional quality control parameters and interpretive criteria for testing susceptibility ofStreptococcus pneumoniae andHaemophilus influenzae to quinupristin/dalfopristin (RP59500)

Studies were undertaken to select tentative criteria for susceptibility testing of quinupristin/dalfopristin againstStreptococcus pneumoniae andHaemophilus influenzae. Against 612 isolates ofStreptococcus pneumoniae, MICs of quinupristin/dalfopristin were 1.0 g/ml for all but one strain. With a tentative MIC breakpoint of either 1.0 g/ml or 2.0 g/ml for susceptible, a disk diffusion zone diameter breakpoint of 19 mm embraced all but two of the susceptible pneumococci; 16 mm included all strains. ForHaemophilus influenzae, MICs of quinupristin/dalfopristin clustered near the tentative breakpoints; 91.5% of the MICs were 2.0 to 8.0 g/ml. This precluded satisfactory performance of the disk diffusion test in discriminating between resistant and susceptible isolates unless MIC breakpoints are modified for this species: clinical experience will be needed before that can be justified. Based on data from a multilaboratory study, the following quality control limits are proposed forStreptococcus pneumoniae ATCC 49619 when testing quinupristin/dalfopristin: 0.25 to 1.0 g/ml for broth microdilution tests and 19 to 24 mm for disk diffusion tests. For tests ofHaemophilus influenzae ATCC 29247, MIC limits are 2.0 to 16 g/ml; disk tests were very reproducible but are not yet recommended.     

In vitro activity of the aryl-fluoroquinolones A-56619 and A-56620 and evaluation of disk susceptibility tests

The activity of two new quinolones, A-56619 and A-56620, was compared in vitro to that of norfloxacin and ciprofloxacin against 6,699 bacterial isolates in four separate clinical laboratories. The overall percentage of strains susceptible to designated concentrations were as follows: 99.1% for norfloxacin (MIC4.0 g/ml), 96.1% for ciprofloxacin (MIC1.0 g/ml), 96.8% for A-56620 (MIC 2.0 g/ml) and 96.1% for A-56619 (MIC 4.0 g/ml). For disk diffusion susceptibility tests 10 g A-56619 disks are tentatively recommended with interpretive standards of 18mm for susceptibility and 13mm for resistance; 5 g A-56620 disks may be used with tentative standards of 19mm for susceptibility and 14mm for resistance.     

Comparison of disk diffusion,the E test,and detection ofmecA for determination of methicillin resistance in coagulase-negative staphylococci

The aim of this study was to find a reliable, fast, and simple alternative to the methicillin disk method for determination of methicillin resistance in coagulase-negative staphylococci, since results of this method are often difficult to read due to growth within the zone of inhibition. The sensitivity of 319 strains of coagulase-negative Staphylococci to a 5 g methicillin disk on Mueller-Hinton agar using an incubation period of 48 h was compared with that of 1 (1 g and 5 g oxacillin disks on Mueller-Hinton agar with or without 2% NaCl, using an incubation period of 24 h. The detection ofmecA (MecAgen) by the polymerase chain reaction was used as a standard. Minimum inhibitory concentrations were determined by means of the E test. Of the 225mecA-positive strains, 190, 215, and 193 were resistant to 5 g methicillin, 1 g oxacillin and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 216, 218, and 223 were resistant on Mueller-Hinton agar with 2% NaCl. Of the 94mecA-negative strains, 89, 93, and 94 were susceptible to 5 g methicillin, 1 g oxacillin, and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 92, 93, and 94 were susceptible on Mueller-Hinton agar with 2% NaCl. Using breakpoints of 2 g/ml for oxacillin resistance and 8 g/ml for methicillin resistance, the E test yielded sensitivities of 99.6 and 99.1% and specificities of 97.9 and 98.9% after 48 h of incubation. The 5 g oxacillin disk was faster and easier to read than the methicillin disk and correlated better with detection ofmecA than the methicillin disk or the 1 g oxacillin disk.     

Effects of PAF and PAF antagonists on the shape of venous endothelial cellsin vitro

Three platelet-activating factor (PAF) antagonists were tested for their ability to prevent or reduce PAF-induced shape changes of large vein endothelial cellsin vitro. BN52021 had a significant protective action at concentrations of 1 M and 0.1 M, but at 100 M had a damaging effect of its own. CV3988 (0.1 M and 1 M) and L652, 731 (20 M) did not reduce the responses to PAF, and at higher concentrations (CV3988 10 M and 100 M, L652, 731 100 M) both compounds alone caused significant changes of shape. BN52021 (0.1 M) was also effective against leukotriene (LT) C₄, at 1 M against bradykinin and LTE₄, and at 10 M against LTD₄ and the calcium ionophore A23187. BN52021 (10 M) was ineffective against shape changes induced by histamine, prostaglandin (PG) E₂ and lysophosphatidylcholine (LPC). Neither indomethacin (100 M) nor verapamil (20 M) altered the response to PAF.Using electron spin resonance (ESR) spectrometry it was shown that the damaging effects of LPC and CV3988 may be due partly to their detergent properties. It is suggested that the mechanism by which PAF alters the shape of large vein endothelial cells is primarily receptor mediated.     

In vitro activity of voriconazole and other antifungal agents against clinical isolates of <Emphasis Type="Italic">Candida glabrata</Emphasis> and <Emphasis Type="Italic">Candida krusei</Emphasis>

The antifungal susceptibility of 309 Candida glabrata and 63 Candida krusei clinical isolates was tested via the Sensititre YeastOne-3 system (Trek Diagnostic Systems, East Grinstead, UK) to compare the in vitro activity of voriconazole with that of five other antifungal agents (amphotericin B, fluconazole, itraconazole, ketoconazole, and flucytosine). Voriconazole was highly active (MIC90, 0.5 g/ml) against isolates of both species, including those for which the MICs of itraconazole and fluconazole were high (MIC90s of itraconazole, 2 g/ml for C. glabrata and 0.5 g/ml for C. krusei; MIC90s of fluconazole, 32 g/ml for C. glabrata and 64 g/ml for C. krusei). Ketoconazole MIC90 values for both species were identical to those of voriconazole. The MIC90 of amphotericin B was similar for both species (0.125 g/ml for C. glabrata and 0.25 g/ml for C. krusei). As expected, flucytosine was only moderately active against C. krusei isolates (MIC90, 16 g/ml) but was highly active against C. glabrata isolates (MIC90, 0.03 g/ml). Potential cross-resistance within the azole class was noted for some strains of C. glabrata (5.5%) that presented high MIC values for all the azoles tested. In order to consider voriconazole a viable alternative to other triazoles for the treatment of infections caused by Candida species, susceptibility testing of all clinically significant isolates of C. glabrata and C. krusei is recommended because of the potential for azole cross-resistance. The Sensititre YeastOne-3 seems to be a suitable commercial tool for this purpose.     

Criteria for testing the susceptibility ofStreptococcus pneumoniae to cefotaxime and its desacetyl metabolite using 1 μg or 30 μg cefotaxime disks

In vitro susceptibility tests were performed with 350 selected strains ofStreptococcus pneumoniae to evaluate disk diffusion tests with 30 g and 1 g cefotaxime disks. Zones were compared to MICs of cefotaxime with and without its desacetyl metabolite. Cefotaxime was two to eight times more active than desacetyl cefotaxime, but the two compounds were additive when combined in vitro. For 30 g disks, zone size breakpoints were 27 mm, 28–30 mm and 31 mm for resistant, intermediate and susceptible, respectively. For 1 g disks, those zone size criteria were reduced to 13 mm, 14–16 mm and 17 mm. The 30 g disk that is currently available for testing other species can be used for testing pneumococci; however, the 1 g disk has some important advantages.     

In vitro activity of LY146032 (Daptomycin), a new peptolide

The in vitro activity of LY146032, a new peptolide antibiotic, was compared with those of vancomycin, teicoplanin, imipenem, amoxicillin and erythromycin. LY146032 inhibited 90% ofStaphylococcus aureus andStaphylococcus epidermidis, including methicillin-resistant isolates at 1g/ml. Its activity was comparableto those of vancomycin and teicoplanin. MIC90s for the beta-hemolytic streptococci varied from 0.25 g/ml for group B streptococci to 4g/ml for some group C and F streptococci. MICs forStreptococcus faecalis were in the range of 0.5 to 8 g/ml,and the MIC90 4 g/ml,compared to 4 g/ml for vancomycin and 1g/ml for teicoplanin. For some viridans streptococci the MICs were 4g/ml, whereasStreptococcus pneumoniae were inhibited by 0. 5g/ml.Corynebacterium JK species were inhibited by 0. 5g/ml, similar to vancomycin, andListeria monocytogenes by 4g/ml.Neisseria species,Haemophilus species and enteric species were not inhibited. Most MBCs were within two-fold of the respective MICs. After 14 days passage in subinhibitory concentrations of LY146032,Staphylococcus aureus, Staphylococcus epidermidis andStreptococcus faecalis showed minimal increase in MICs. The activity of LY 146032 was increased by adding Ca²⁺ and was reduced in an anaerobic environment. Overall, LY146032 is an extremely interesting new agent that inhibits gram-positive species.     

Effect of praziquantel on the free-living stages ofSchistosoma mansoni

Summary The effect of the new schistosomicide praziquantel (2-cyclohexyl-carbonyl-1,2,3,6,7,11b-hexahydro-2H-pyrazino[2,1a]isoquinolin-4-one) on the miracidia and cercariae ofSchistosoma mansoni was investigated.In vivo praziquantel inhibits hatching of miracidia for 24 h after administration of 500 mg/kg to infected mice. In vitro a concentration of 10 g/ml inhibits subsequent hatching in drug-free water. Free swimming miracidia are rapidly killed by 1 g/ml. Even 0.01 g/ml is still partially effective.In a solution of 0.03 g/ml cercariae lose their ability to swim within 10 min. This effect is reversible in drug-free water. Morphological damage to cercariae incubated in 0.1 g/ml is clearly evident. However, cercariae are fully infective when given subcutaneously to mice after a 3-h incubation period. Incubation in 1 g/ml reduces the infection rate by 80%. A 2-h incubation in 0.1 g/ml almost completely inhibits the percutaneous infection through the abdominal skin. The number of cercariae that develop to schistosomules is reduced by more than 90%. After a 2-h incubation in a concentration of 0.01 g/ml the swimming ability of cercariae is impaired in such a way that the number of cercariae penetrating in the tail immersion test and developing to schistosomules is reduced by half.Praziquantel is a more potent protective agent than the molluscicides copper sulphate, sodium pentachlorophenate and Bayluscide^® or cadmium and zinc ions.

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Zusammenfassung Es wurde der Einfluß des neuen Schistosomenmittels Praziquantel (2-Cyclohexylcarbonyl-1,2,3,6,7,11b-hexahydro-2H-pyrazino[2,1 a]isochinolin-4-on) auf die Miracidien und Cercarien vonSchistosoma mansoni untersucht.In vivo verhindert Praziquatel das Schlüpfen von Miracidien aus Eiern für 24 h nach einer Behandlung infizierter Mäuse mit 500 mg/kg. Nach Inkubation von Eiern in 10 g/ml schlüpfen anschließend keine Miracidien in präparatfreiem Wasser. Frei schwimmende Miracidien werden durch 1 g/ml schnell abgetötet und noch 0,01 g/ml haben eine deutliche Wirkung.Cercarien werden in vitro durch 0,03 g/ml innerhalb von 10 min schwimmunfähig. Dieser Effekt ist in präparatfreiem Wasser reversibel. Nach 3stündiger Vorbehandlung in 0,1 g/ml sind die Cercarien deutlich morphologisch geschädigt. Trotzdem sind sie noch normal entwicklungsfähig, wenn sie Mäusen subcutan injiziert werden. Nach Inkubation in 1 g/ml ist der Infektionserfolg allerdings um 80% reduziert. Nach nur 2stündiger Inkubation in 0,1 g/ml ist die Zahl der Cercarien, die sich nach percutaner Infektion durch die Bauchhaut der Maus zu Schistosomulae entwickelt, um mehr als 90% reduziert. Nach 2stündiger Inkubation in nur 0,01 g/ml ist das Schwimmvermögen der Cercarien so beeinträchtigt, daß sich nach percutaner Schwanzinfektion nur noch halb so viele zu Schistosomulae entwickeln wie in der Kontrolle.Praziquantel ist in seiner infektionsverhindernden Wirkung den Molluskiziden Kupfersulfat, Pentachlorphenolnatrium und Bayluscid^® sowie auch Cadmium- und Zinkionen überlegen.

     

Activity of trimethoprim/ sulfamethoxazole plus polymyxin B against multiresistantStenotrophomonas maltophilia

The inhibitory activity of eight antibiotics and the inhibitory and bactericidal activities of combinations of trimethoprim/sulfamethoxazole (TMP/SMX) plus three fixed concentrations of polymyxin B (0.01 g/ml, 0.1 g/ml and 0.5 g/ml) against 30 multiresistant strains ofStenotrophomonas maltophilia were tested. Polymyxin B at 0.01 g/ml modified the inhibitory activity of TMP/SMX against only 40% of strains. At 0.1 g/ml and 0.5 g/ml, polymyxin B enhanced the inhibitory activity of TMP/SMX activity against all strains. Polymyxin B enhanced the bactericidal activity of TMP/SMX only at concentrations near the minimum inhibitory concentration of polymyxin B alone.     

High-resolution chromosome R-banding in lymphoblastoid cell lines by the combined use of cell synchronization and ethidium bromide treatment

Summary A reliable method for obtaining high-resolution R-banded chromosomes from lymphoblastoid cell lines is described. The cell cultures are subjected to S-phase synchronization in the presence of excess thymidine (300 g/ml) for 17 to 19 hr, followed by BrdU treatment (30 g/ml) for 6.5 hr. Prior to harvest, they are exposed to ethidium bromide (7.5 g/ml) for 1.5 hr and Colcemid (0.02 g/ml) for 30 min. Using this method, high-resolution R-banded chromosomes at the 550–850 band level were obtained with frequencies at high as 70% of all mitotic cells.     

Susceptibility testing quality control studies with fosfomycin tromethamine

Multiple studies were performed to define the quality control ranges for susceptibility tests of fosfomycin tromethamine. For disk diffusion tests, the limits proposed are 22 to 30 mm forEscherichia coli ATCC 25922 and 25 to 33 mm forStaphylococcus aureus ATCC 25923. Broth microdilution tests were not reproducible. For agar dilution tests, ranges proposed are 0.5 to 2.0 g/ml forEscherichia coli ATCC 25922, 0.5 to 4.0 g/ml forStaphylococcus aureus ATCC 29213; 2.0 to 8.0 g/ml forPseudomonas aeruginosa ATCC 27853, and 32 to 128 g/ml forEnterococcus faecalis ATCC 29212.     

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold on the cell surface and whose CH1 sequence was removed to prevent from being retained in the endoplasmic reticulum. It efficiently and stably expressed chains of IgM on the cell surface ( _m ⁺ ) without light chains. To obtain mutants lacking _m ( _m ^– ) from the _m ⁺ cell line by selectively killing _m ⁺ cells, a method with ricin A-conjugated anti- antibody was more reliable than complement lysis mediated by anti- antibody. Applying the system, we obtained a variety of _m ^– mutants.     

Control of recombination within and between DNA plasmids of Saccharomyces cerevisiae

Summary [2 m⁺ and [2m°] yeast were transformed to stable leucine prototrophy with the hybrid yeast — E. coli plasmid, pJDB219. This plasmid contains the entire sequence of the endogenous 2 m yeast DNA plasmid in addition to the yeast nuclear LEU2 ⁺ gene and the Co1E1 derivative, pMB9. In the [2 m⁺] transformants, a new wholly yeast LEU2 ⁺ plasmid, pYX, was generated, probably by a recombination event between pJDB219 and 2 m DNA. The plamid, pYX, in the absence of 2 m DNA, was found to exist in equimolar amounts of two forms, A and B, which probably arise by intramolecular recombination across the inverted repeat sequences of the 2 m DNA portion of the plasmid. pJDB219 was found to require the presence of 2 m DNA to undergo this intramolecular recombination. The results suggest that 2, m DNA and pYX code for a gene product required in this recombination event which pJDB219 cannot produce.     

Trypanosoma congolense: the in vitro akinetoplastic induction sensitivity assay

Incubation ofTrypanosoma conglense in diminazene aceturate (Berenil) or isometamidium chloride (Samorin) induced akinetoplastic (AK) forms in vitro. The AK values (expressed in percent) obtained were found to be useful for rapid assessment of relative drug sensitivities. In susceptible clones, AK forms were induced at all drug concentrations tested, whereas in resistant clones they were induced only at higher concentrations. The Berenil-resistant clone exhibited AK values of 0.9%±0.6%–8.9±2% at concentrations of 1–100 g/ml at 4–10 h post-inoculation (p.i.), whereas the Berenil-susceptible clone displayed values of 9.3%±3%–19.2%±5% at 0.1–50 g/ml. Motile trypanosomes were not seen at 100 g/ml at 4 h p.i. or at 10 or 50 g/ml at 10 h p.i. The Samorin-resistant clone showed AK values of 0.5%±0.1%–43%±3% at concentrations of 0.1–100 g/ml at 4 and 10 h p.i., whereas the Samorin-susceptible clone exhibited values of 5.3%±2%–45%±4% at 0.0005–100 g/ml. These results were supported by the findings obtained using a mouse infectivity test.     

2 μm plasmid copy number in different yeast strains and repartition of endogenous and 2 μm chimeric plasmids in transformed strains

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 m plasmid in yeast cells. For this purpose we determined the 2 m plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 m chimeric plasmids.According to the strain used the proportion of 2m DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 m molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 m copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 m DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 m copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 m DNA copies per haploid genome is transformed with 2 m chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 m chimeric plasmids were introduced in our 2 m-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.     

Lactoferrin,lysozyme, and β2-Microglobulin levels in cerebrospinal fluid

The CSF levels of lactoferrin, lysozyme, and ₂-microglobulin ( ₂ ) were measured in patients with evident, probable, or possible inflammatory CNS reactions and compared to those found in neurologically apparently healthy patients. Patients with viral CNS infections had significantly raised ₂ and lysozyme levels but normal lactoferrin levels, indicating a local activation of lymphocytes and monocytes but not of granulocytes. Patients with bacterial CNS infections had significantly raised levels of all three cell markers, but the increase of lysozyme and lactoferrin was relatively more pronounced than that of ₂ , indicating that the inflammatory response to bacterial agents is dominated by monocytes and granulocytes. Patients with primary or secondary malignant brain tumors were characterized by a moderate increase of ₂ and a considerable increase in both lysozyme and lactoferrin, i.e., the same protein pattern as observed in bacterial CNS infection. The lysozyme levels were moderately increased in half the patients with benign cerebral tumors while the levels of ₂ and lactoferrin were normal, indicating that benign and malignant brain tumors induce different local inflammatory CNS reactions. Half the patients with pituitary gland adenoma had elevated ₂ and lysozyme levels but normal lactoferrin levels, suggesting that immunological mechanisms are associated with the adenoma development. Patients with MS had moderately but significantly raised CSF levels of ₂ and lysozyme and a third of them also had raised levels of lactoferrin, a protein pattern suggesting a low-active inflammatory process in CNS involving mononuclears and granulocytes. A similar protein pattern was found in Guillain-Barré syndrome. In cerebrosarcoidosis we noted considerably increased lysozyme and ₂ but normal lactoferrin levels, consistent with the idea that the sarcoid granuloma mass is dominated by monocytic inflammatory cells. The data obtained indicate a clinical value of lactoferrin, lysozyme, and ₂ as differential indices of inflammatory cell reactions taking place in various CNS processes.     

Expression of surface membrane IgG on pokeweed mitogen-reactive anti-tetanus toxoid antibody-producing cells

Anti-tetanus toxoid antibody-producing cells, differentially expressing surface membrane IgM, were analyzed for the additional expression of surface membrane IgG. ⁺ and ^– cells were rosetted with anti--ox red blood cells and separated by density centrifugation into fractions enriched or depleted or ⁺ cells. These B-cell subsets were assayed for the production of IgM and IgG anti-tetanus toxoid antibody and total IgM and IgG. The results indicated that the majority of anti-tetanus toxoid antibody synthesis in the ^– fraction was by ⁺ cells. In the ⁺ fraction, however, both IgM and IgG anti-tetanus toxoid antibody production was detected in the ^+– and ⁺⁺ fraction. The inclusion of isotype-specific antisera during the first 2 days of culture further established that was expressed on the surface of the majority of the precursors for IgG anti-tetanus antibody productionin vitro. Studies performed to determine the culture requirements of ^– and ⁺ cells revealed that production of IgG anti-tetanus toxoid antibody by both cell subsets was dependent on T cells and pokeweed mitogen. However, some ^– cells could produce IgG in the presence of T cells alone.     

Inhibitory effect of phloretin on histamine release from isolated rat mast cells

The ability of the flavonoid phloretin to inhibit histamine release from rat mast cells varied considerably with the releasing agent investigated. The response to the combination of the ionophore A23187 and the phorbol ester TPA and to suboptimal concentrations of the ionophore (0.5 M) was potently inhibited (IC₅₀ about 5 M), whereas phloretin was less potent against responses to the ionophore (1 M) IC₅₀ of 17 M), to antigen alone and in combination with TPA (IC₅₀ of 30–50 M), to TPA in the absence of calcium (IC₅₀ of 50 M) and to compound 48/80 in the absence and presence of calcium (IC₅₀ of 60–90 M). The inhibition by phloretin at concentrations above 10M was partly counteracted by glucose (5 mM) indicating effects on oxidative metabolism. The flavonoid quercetin was equally potent in inhibiting histamine release induced by antigen, the ionophore at different concentrations and in combination with TPA (IC₅₀ of 20M). Although not conclusive, the results are consistent with an inhibition of protein kinase C by phloretin at concentrations below 10 M. At higher concentrations unspecific actions become apparent and phloretin therefore seems to be of limited utility as a probe for signal-pathways in cell responses.     

Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca²⁺-dependent K⁺ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (I _oo). Neomycin (30 M), an inhibitor of phospholipase C, and intracellular applications of heparin (10 g/ml) and guanosine 5-O-(2-thiodiphosphate) (GDP[S]; 1 mM) partly but consistently inhibited the generation of I _oo, whereas a higher concentration of heparin (100 g/ml) transiently enhanced then suppressed the generation of I _oo. Inhibition of I _oo generation by heparin was more powerful at the holding potential of + 20 mV than at –20 mV. Inositol 1,4,5-trisphosphate (InsP ₃; 30 M) continuously generated I _oo at holding potentials more positive than –60 mV. Noradrenaline (10 M) and caffeine (3–20 mM) transiently augmented, then reduced the generation of I _oo. Heparin (10 g/ml) completely inhibited responses induced by InsP ₃ and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5-triphosphate (GTP; 200 M) or low concentrations of guanosine 5-O-(3-thiotriphosphate) (GTP[S]; 3 M) continuously augmented the generation of I _oo. High concentrations of GTP[S] (10 M) transiently augmented, then inhibited I _oo. Neither GTP[S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I _oo when applied in the presence of GDP[S] (1 mM), neomycin (30 M) or heparin (10 g/ml). PDBu (0.1 M) reduced the generation of I _oo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 M). In the presence of H-7, GTP[S] continuously enhanced the generation of I _oo. The suppression of the generation of I _oo during application of noradrenaline (10 M) was reduced by pretreatment with H-7. Thus both InsP₃ and protein kinase C contribute to the generation of I _oo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP ₃ antagonist on the InsP ₃-induced Ca²⁺-release channel (PIRC). InsP ₃ opens PIRC and protein kinase C may deplete the stored Ca²⁺ by either inhibiting the reuptake of Ca²⁺ or by enhancement of the releasing actions of InsP ₃.